Fluorescence microscopy is widely used in molecular and cell biology and other applications for non-invasive, time-resolved imaging. Despite these advantages, standard fluorescence microscopy is not useful for ultra-structural imaging, due to the optical diffraction limit. The optical diffraction limit is a physical property that limits the resolution of conventional microscope systems. Due to its wave properties, light passing through a circular lens creates a ring-shaped diffraction pattern; the images of two different points formed by such a lens can be resolved if the principal diffraction maximum of one point lies outside of the first minimum of the other point. This theoretical diffraction limit, is approximately equal to 0.61·λ/NA, where λ is the wavelength of the light and NA is the numerical aperture of the lens, given byNA=n·sin α  (Formula I)where n is the index of refraction of the optical medium between the lens and the specimen and α is the half-angle of acceptance of the lens. Currently available microscope objective lenses typically have NA<1.4, so that the theoretical diffraction limit for visible light is >200 nm; in practice the resolution limit of standard optical microscopes, compromised by various lens aberrations, is poorer, seldom much below 500 nm.
Over the past several years, a number of new technical innovations have been introduced that effectively circumvent the optical diffraction limit, opening the door to vastly improved fluorescence microscopy image resolution. The first of these techniques, termed Stimulated Emission Depletion (“STED”) microscopy, relies on a fluorescence excitation source coupled to a second illumination beam that prevents fluorescence relaxation of all but a small volume of fluorophores in the sample, thereby greatly enhancing image resolution. The second depletion beam used in STED acts to force an excited fluorophore to the ground state by stimulated emission. As such, a spectrally distinct signature is produced that is separable from the fluorescence emitted from molecules that have not undergone the depletion process.
In order to achieve spatial separation between the two processes, the second depletion beam is phase-shaped such that it produces an optical vortex at the sample—rendering a very small (typically 30-50 nm across) point at the center of the vortex that is sufficiently free of depletion energy. This “depletion-free” zone allows only a small, sub-diffraction volume of fluorophores to be detected. Subsequent raster scanning of the coupled excitation/depletion beams can then be used to assemble an image with greatly enhanced detail.
While representing a monumental advance in the field of molecular imaging, STED microscopy retains some important limitations. Firstly, the system complexity and cost is formidable. Original STED systems consisted of two mode locked femtosecond pulsed laser sources that required sophisticated synchronization in order to assure that the excitation and depletion laser pulses reached the sample within less than 100 picoseconds of each other. The introduction of a super-continuum based STED system has reduced this burden. However, most STED imaging systems still rely on a single excitation and single depletion beam. This generally limits STED to the detection of a single fluorophore in the sample. While multi-color STED systems have been demonstrated, they require the use of multiple (and synchronized) pulsed laser sources as described above, thereby preventing practical implementation in nearly any biological research settings.
Accordingly, new techniques capable of multiplexed detection are needed to harness the benefits of fluorescence microscopy for ultra-resolution imaging of biological and other samples.